Pharmaceutical composition containing 2-dimethylamino-6-diethyleneimidophosphamido-7-methyl-purine

ABSTRACT

2-Dimethylamino-6-diethyleneimidophosphamido-7-methylpurine having the formula ##STR1## A method for preparing 2-dimethylamino-6-diethyleneimido-phosphamido-7-methylpurine consisting of treating theo-bromine with heating to the boiling point and subsequently with phosphorus oxychloride and phosphorus pentachloride. The prepared 2,6-dichloro-7-methylpurine is heated to 50° - 60° C in an aqueous solution of ammonia and the resulting 2-chloro-6-amino-7-methylpurine is reacted, with heating, with a aqueous solution of dimethylamine. The obtained 2-dimethylamino-6-amino-7-methylpurine is reacted, at the boiling point, with either phosphorus oxychloride, or phosphorus pentachloride and formic acid. The resulting 2-dimethylamino-7-methylpurinyl-6-aminophosphoric dichloroanhydride is then treated with ethyleneimine in a medium of an organic solvent in the presence of an acceptor of hydrogen chloride, and the final product is then isolated. 
     A preparation for treating malignant tumors of the haemopoietic system, containing 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine as the active principle is provided.

This is a continuation of application Ser. No. 654,772, filed Feb. 3, 1976 which in turn is a Continuation of U.S. Ser. No. 538,521, filed Jan. 6, 1975, which in turn is a continuation of U.S. Ser. No. 339,346, Mar. 8, 1973, all now abandoned.

The present invention relates to a new substance, namely 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine, and a method for preparing same and to a medicinal preparation based on this substance.

The said substance, according to the invention, has the following formula: ##STR2##

The proposed new compound is a colourless substance, soluble in water to a limited extent, readily soluble in chloroform, and also in boiling alcohol (1:5), toluene (1:70), benzene (1:70) and dichloroethane (1:6).

The substance has no definite melting point and is decomposed gradually at temperatures above 250° C.

2-Dimethylamino-6-diethyleneimidophosphamido-7-methylpurine has cancerolytic activity and can therefore be used in medicine as a preparation against malignant growths in the haemopoietic system.

Preparations are known in the prior art which are used for the said purposes, for example, thiophosphamide, dipin, and others, the cancerolytic properties of which are due to the presence of ethyleneimine groups.

However, these preparations have insufficient selectivity for their action on tumour tissue. Their anti-tumoric effect is only transient and can be observed only with doses that produce side effects.

According to the invention, the method for preparing 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine consists heating the obromine to the boiling point, and subsequently treating same with phosphorus oxychloride and phosphorus pentachloride, thus forming 2,6-dichloro-7-methylpurine which is heated to from 50° to 60° C. in an aqueous solution of ammonia, and the resulting 2-chloro-6-amino-7-methylpurine is heated and acted upon by an aqueous solution of dimethylamine, and the thus formed 2-dimethylamino-6-amino-7-methylpurine is then reacted with phosphorus oxychloride or phosphorus pentachloride and formic acid at the boiling point temperature, with subsequent processing of thus formed 2-dimethylamino-7-methylpurinyl-6-amidophosphoric dichloroanhydride with ethyleneimine in an organic solvent medium in the presence of a hydrogen chloride acceptor, and then isolating of the final product.

Chloroform, benzene, dichloroethane and ethylene chloride are used as the organic solvent in isolating the final product.

As was mentioned above, 2-dimethyl amino-6-diethyleneimidophosphamido-7-methylpurine have cancerolytic activity and can be used as the active principle of a new pharmaceutical preparation useful in the treatment of acute leukemia.

The new preparation is given the name `phopurine`.

Phopurine can be given to patients in the powder form (lyophilized on a 5 percent vinylpyrrolidone). When given intrevenously or intramuscularly, it is recommended that the preparation containing the said active principle be used with a pharmaceutical solvent or carrier.

An isotonic solution of sodium chloride or distilled water can be used as the pharmaceutical solvent for the new preparation.

It is recommended that a from 0.1 to 1 percent solution of the new preparation in a pharmaceutical solvent be used.

The injection solution should be prepared just before injection by dissolving the sterile powder of 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine in distilled water or in an isotonic solution of sodium chloride.

While studying the anti-tumour activity of the new preparation on animals, we established that it possesses a remarkable power to inhibit growth of many tumours grafted on rats and mice. Jensen's sarcoma strain appeared to be the most sensitive towards the action of phopurine. The administration of a single dose of 60 mg per kg body weight inhibited the growth of the tumour 95 percent.

A marked anti-leukemic activity of phopurine was noted in mice of the C₅₇ line with transplanted La leukemia. In all experiments, the animals to which the preparation was given, lived 2-3 times longer than the untreated animals.

Phopurine is superior to many other derivatives of ethyleneimine, e.g. thiophosphamide, dipin, etc., with respect to its anti-tumour activity.

The data on the anti-tumoric effect of phopurine, thiophosphamide and dipin on the life duration of mice with La leukemia are given in the Table below

    ______________________________________                                         Preparation  Maximum duration of life, in % to control                         ______________________________________                                         Thiophosphamide                                                                             20                                                                Dipin        56                                                                Phopurine    204                                                               ______________________________________                                    

The haematological indices also show the favourable course of the transplanted La lukemia in mice which underwent therapy with the preparation. The total quantity of leukocytes in mice with La leukemia almost did not differ from the starting counts in the end of the experiment with the phopurine therapy, while in control mice with leukemia, the leukocyte count increased significantly by the end of life. The quantity of immature forms in the controls increased sharply by the end of the experiment, while in the phopurine emperimental animals this increase was insignificant.

Unlike the other derivatives of ethyleneimine, phopurine does not produce profound leukopenia in animals treated with the preparation.

It has been established that phopurine is less toxic than thiophosphamide or dipin. The cumulative properties of phopurine are less pronounced: the cumulative index of phopurine are less pronounced: the cumulative index of phopurine is 4 times less than in thiophosphamide and 7 times less than in dipin.

With respect to the character of its action on the biological systems, phopurine refers to alkylating cytostatic preparations. When given in small doses, the preparation selectively inhibits the proliferating processes in tissues, including tumour tissues. Like other alkylating substances, phopurine reacts with the nucleic centres of nucleic acids molecules and protein and alters various cell functions. The administration of the preparation in large tolerated doses produces a more significant destroying action on the tumour cells as compared with the action on the intact tissue cells, which is the essence of the anti-tumoric action of the proposed preparation.

The new preparation was tested clinically on 80 patients with chronic myeloleukemia and acute leukemia. The preparation was quite effective in treating patients with chronic myeloleukemia in which the known anti-tumour preparations (myelosan, myelobromol) proved ineffective.

In treating patients with chronic myeloleukemia the preparation should be given first in doses of 40 mg (0.1-1.0 percent solution in a pharmaceutical solvent) daily. If it is tolerated well (6-7 intramuscular injections), the dose may be increased gradually to from 60 to 80 mg. (A thorough control of the blood should be effected during this therapy). The total dose of the preparation should be from 1.5 to 3 g.

Under the action of the phopurine therapy in patients with chronic myeloleukemia, the spleen diminished in size, and the leukocyte count decreased from 30 to 80 percent, with a simultaneous decrease in the number of immature forms of the myeloid series.

The efficacy of treating chronic myeloleukemia depends on the extent to which the haemopoietic organs are affected and also on the dose of the preparation given during the entire course of the treatment.

The preparation is well tolerated by the patients. A possible complication due to overdosage or hypersensitivity to the preparation is leukopenia, for which reason the blood should be regularly analyzed during the enitre course of the therapy as well as within two weeks following the suspension of the therapy.

The new preparation is contraindicated to patients with grave diseases of the liver and kidneys, and also to patients in terminal states, and with marked leukopenia.

Phopurine is dispensed in 0.04 g doses in sterile vials. The preparation should be stored in cool dark premises. The expiration term is one year (and over).

The method for preparing 2-dimethylamino-6-diethyleneimidophosphamide-7-methylpurine, which is the active principle of the preparation phopurine, should be effected preferably as follows:

Theobromine is boiled with excess phosphorus oxychloride until a solution is formed. Phosphorus pentachloride is added thereto and the resultant solution is boiled for from 10 to 12 hours in order to ensure its complete dissolution. The reaction mass is then cooled, passed through a filter, and the separated precipitate (without drying or purification) is transferred with stirring into a cooled (5°-10° C.) aqueous solution of ammonia and then heated to from 50° to 60° C. in the presence of n-butyl alcohol (to prevent foaming) for from 10 to 12 hours. The thus formed product is separated and (without purifying) heated with an excess of a 33 percent aqueous solution of dimethylamine with a subsequent cooling and filtering.

The prepared 2-dimethylamino-6-amino-7-methylpurine is purified by recrystallization from an aqueous alcohol solution and the purified product is then phosphorylated by two methods, either by phosphorus oxychloride or by phosphorus pentachloride and formic acid. In the former case, it is boiled with an excess quantity of phosphorus oxychloride for 8 hours, then phosphorus oxychloride is removed by vacuum distillation at 45°-50° C. and the crystalline precipitate is mixed thoroughly with dry toluene which is then removed (together with residual POCl₃) by vacuum distillation.

According to the other method, 2-dimethyl-amino-6-amino-7-methylpurine is boiled in chloroform with a pre-calculated quantity of phosphorus pentachloride for about 5 hours until the hydrogen chloride stops evolving; the solution is then cooled and a pre-calculated quantity of formic acid is added slowly with stirring at a temperature of 20° C. The mixture is boiled for one hour, and chloroform and hydrogen chloride are vacuum distilled.

An organic solvent, preferably chloroform, is added to the product prepared by either method, and then a mixture of ethyleneimine and potassium carbonate (aqueous solution) is added slowly to the resultant suspension. The reaction mixture is stirred for 30 minutes at 20° C., and the chloroform layer is separated, dried and evaporated in a vacuum at a temperature of about 35° C. The residue is purified by crystallization first from alcohol and then from toluene, and the pure 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine is finally prepared.

Sodium bicarbonate, triethylamine, etc, can also be used as acceptors of the hydrogen chloride (instead of potassium carbonate).

For a better understanding of the invention, the following examples of preparing 2-dimethylamino-6-diethyleneimidophosphamide-7-methylpurine are given hereinbelow by way of illustration.

EXAMPLE 1.

(a) 200 g of theobromine are boiled with 1000 ml of phosphorus oxychloride for from 9 to 10 hours, and cooled, and boiled again in solution with 464 g of phosphorus pentachloride for another from 10 to 12 hours. The precipitate and is separated and transferred immediately into a mixture of 2 liters of a 25 percent solution of ammonia in water and 1 kg of ice, and then heated to from 50° to 60° C. for 10-12 hours in the presence of 60 ml of n-butyl alcohol, and the reaction mixture is passed through a filter, and the precipitate washed with water in order to prepare 218 g (146,5 g as dry product) of 2-chloro-6-amino-7-methylpurine.

The phosphorus oxychloride is removed from the mother liquor by distillation, and used in the next cycle without any purification. The still residue contains about 5 percent of 2-chloro-6-amino-7-methylpurine which should be recovered.

The moist 2-chloro-6-amino-7-methylpurine (218 g) prepared in mixed with 1500 ml of a 33 percent aqueous solution of dimethylamine and stirred at 65°-75° C. for 5-6 hours. The dimethylamine solution is distilled off (about 600 ml), and the residue cooled and passed through a filter, and crystallized from 1100 ml of a 80 percent alcohol solution. The yield of the 2-dimethylamino-6-amino-7-methylpurine is 96.5 g. The melting point is from 280° to 284° C.

Calculated, in percent: C 50.0; H 6.25; N 43.75; C₈ H₁₂ N. Found, in percent: C 49.65; H 6.01; N 43.78.

(b) 96.5 g of 2-dimethylamino-6-amino-7-methylpurine are boiled with 500 ml of phosphorus oxychloride with stirring for eight hours. The phosphorus oxychloride is distilled, and the residue mixed 600 ml of dry toluene; the toluene is distilled in vacuum, and 2 ml of chloroform is added to the residue and mixed. Now (with cooling to to from 5° to 10° C.) a mixture of 57.9 ml of ethylenimine is added along with a solution of 104 g of potassium carbonate in 520 ml of water to the suspension which is mixed for thirty minutes at 20° C. The chloroform layer is separated, dried, evaporated, and crystallize the residue is crystallized first from alcohol (1:5) and then from toluene (1:70). The yield of the doubly-crystallized 2-dimethyl-amino-6-diethyleneimidophosphamido-7-methylpurine is 31-32 percent.

Calculated, in percent: C 44.72; H 5.90; N 34.78; P 9.62. C₁₂ H₉ N₈ OP. Found, in percent: C 44.69; H 5.78; N 34.69; P 9.57.

EXAMPLE 2.

96.5 g of 2-dimethylamino-6-amino-7-methylpurine prepared by the procedure described in item (a), Example 1, are boiled for 5 hours with 104 g of phosphorus pentachloride in 1000 ml of dry alcohol-free chloroform.

19.3 ml of formic acid are added to the solution at 60° C., and boiled for one hour, after which it is evaporated in a vacuum at 30° to 40° C. 2 liters of chloroform are added to the residue, and then, with energetically stirring, at from 5° to 10° C., add a mixture of 57.9 ml of ethylenimine and 104 g of potassium carbonate solution a in 520 ml of water are added thereto and mixed for thirty minutes. The chloroform is separated from the solution, dried, and the chloroform is distilled at about 35° C. The the residue is crystallized first from alchol (1:5) and then from toluene (1:70). The yield of the doubly crystallized 2-dimethylamino-6-diethyleneimidophosphamido-7-methylpurine is 31-32 percent. 

What we claim is:
 1. A pharmaceutical composition for use in the treatment of acute leukemia, said composition consisting essentially of a therapeutically effective amount of 2-dimethylamino-6-diethylenimidophosphamido-7-methylpurine as the active principle in combination with a pharmaceutical carrier.
 2. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutical carrier is selected from the group consisting of distilled water and an isotonic solution of sodium chloride.
 3. The pharmaceutical composition as claimed in claim 2, wherein the active principle is present in an amount of from 0.1 to 1.0 percent. 